Bacterial species and their role in delaying the healing of pressure ulcers (PU) in spinal cord injury (SCI) patients have not been well described. The aim of this study was to characterize temporal evolution of superficial and deep microbiota of PU in SCI population. Patients treated for PU between 05/2015 and 12/2016 at the Propara Neurological Rehabilitation Center in Montpellier (France) were included. After wound debridement, samples for bacterial cultures were obtained by swabs (superficial) and tissue biopsies (deep) at the day of inclusion (D0) and 28 after (D28). Forty-nine patients were included (median age: 56 years; 79.6% men). At deep level, coagulase-negative staphylococci (33.8% at D0; 20% at D28), S. aureus (17% at D0; 21% at D28), Enterococcus sp. (12,3% at D0; 16% at D28) and Corynebacterium sp. (13.8% at D0; 16% at D28) were the most frequent bacterial species isolated. After 28 days, percentage of Enterobacteriaceae had significantly increased (0% at D0; 10.5% at D28; P < 0.05) and percentage of anaerobic bacteria had significantly decreased (10.7% at D0; 5.3% at D28; P < 0.05). During the follow-up, at the intra-individual level, the bacterial species isolated were significantly different between D0 and D28, especially at the deep level. This great variability was also described between the deep and the superficial levels. This study highlights for the first-time the evolution of the ecology of PU during one month. The low proportion of Gram-negative bacilli suggests that these bacteria are often colonizing bacteria that could be eliminated by an efficient surgical or mechanical debridement. Moreover, this work demonstrates the uselessness of superficial swabs that do not reflect the bacterial ecology present in depth of the wound.